We propose to generate, validate and distribute the second version of the Xenopus ORFeome, which we define as a fully sequenced, validated set of Xenopus cDNA clones containing one each of every open reading frame (ORF) encoded in the genome, in a format in which any ORF sequence can be easily transferred using recombineering into a diverse array of expression vectors. This one set of reagents will greatly decrease the time to characterize any protein in the myriad functional assays carried out by the entire Xenopus community. But most importantly, an ORFeome set will allow high-throughput in vivo functional-genomic screening in manner currently not feasible, which is a particular strength of the Xenopus system where functional screens have been the basis of a number of fundamental biological observations. Our consortium completed the first phase of this project where we moved 90% of the available full-length cDNA clones to Gateway compatible vectors. However, because of limitations of the original Xenopus EST projects this represents only half of all of the ORFs in the genome. In this project we will de novo clone the remaining Xenopus ORFs to a Gateway donor vector to generate the only available cDNA clones for over half the genome. These clones will be made available, without restriction, to researchers worldwide. The Xenopus community is fully supportive of this project and at a recent PI meeting (MBL (Woodshole), September 2015) the ORFeome was voted as the Top priority of needed Xenopus resources.